Method for host protein analysis in recombinant protein preparations

Abstract

This invention relates to an improved sample preparation method for LC-MS / MS analysis, which enables efficient separation, purification, and identification of protein contaminants, such as host cell proteins (HCPs) that are process-related impurities generated in biopharmaceutical processes such as monoclonal antibody (mAb) production.

Description

Technical Field

[0001] This invention relates to an improved sample preparation method for LC-MS / MS analysis, which enables efficient separation, purification, and identification of protein contaminants, such as host cell proteins (HCPs) that are process-related impurities generated in biopharmaceutical processes such as monoclonal antibody (mAb) production. Background Technology

[0002] Host cell proteins (HCPs) are process-related impurities generated during biopharmaceutical manufacturing and can contaminate the final product unless effectively removed. Due to the potential impact of HCPs on product safety and efficacy, regulatory authorities require that HCPs be removed to trace levels in the final manufactured biopharmaceutical. The current standard method for detecting HCPs is enzyme-linked immunosorbent assay (ELISA), which only reveals the total amount of HCPs.

[0003] A sensitive and high-throughput LC-MS technique has proven to be a potential alternative, allowing for the identification and quantification of individual HCPs. However, variations in sample preparation methods and MS acquisition techniques have led to inconsistencies in the HCPs detected across studies, potentially compromising patient safety and efficacy. To address this issue, the inventors have developed a novel and reproducible method for isolating and digesting HCPs for subsequent mass spectrometry detection. This method is applicable to downstream processes of recombinant proteins such as monoclonal antibodies (mAbs) and can be easily implemented in a standard laboratory without the need for specialized equipment.

[0004] Therapeutic monoclonal antibodies (mAbs) are currently the main category of newly approved drugs worldwide, with more than 100 mAbs approved for the treatment of various diseases and about 1,200 undergoing clinical trials.

[0005] Most mAbs are produced in engineered cell lines, particularly mammalian cells, which can introduce unwanted process-related impurities called host cell proteins (HCPs). If these HCPs are not effectively removed, they can induce immunogenic responses in patients and impair product stability by degrading formulation excipients (such as polysorbate) or the mAb itself. Therefore, regulatory authorities, such as the European Medicines Agency (EMEA) and the U.S. Food and Drug Administration (FDA), have mandated that HCPs be extensively removed throughout the manufacturing process. Although specific specifications are lacking, manufacturers typically aim for HCP levels in the final product to range from 1 to 100 parts per million (ppm). Consequently, various analytical techniques have been developed for the routine assessment of ppm levels of HCPs during biopharmaceutical manufacturing.

[0006] Enzyme-linked immunosorbent assay (ELISA) has traditionally been used to monitor hematologic colloids (HCPs) at ppm levels. However, because this method cannot identify individual HCPs, most biopharmaceutical manufacturers implement orthogonal methods to mitigate the risk. Recent advances in liquid chromatography-mass spectrometry (LC-MS) may make bottom-up proteomics a promising tool for HCP assessment. In this method, proteins are enzymatically digested and then identified at the peptide level. Once an HCP is identified, specific removal procedures and risk assessments can be performed to support process development. However, given the high dynamic range (five to six orders of magnitude) of the relative amounts of mAb and HCP in samples from any mAb production process, the success of this strategy is strongly dependent on sample preparation procedures (e.g., peptide fractionation or mAb depletion) and the sensitivity of MS detection. Improvements in sample preparation methods are needed to address this challenge and lower the detection limits for HCPs.

[0007] Inconsistencies in the identification of HCPs across studies, due to differences in the workflows used, can pose risks to patient safety and drug efficacy. For example, in multiple studies, the number of HCPs identified from NIST mAbs ranged from 60 to 850 proteins. The diversity of HCPs identified in therapeutics is even more important when such data is used to guide manufacturing processes and assess the safety of the final drug for widespread use in the population.

[0008] One of the major sources of variability in identifying HCPs via bottom-up proteomics is sample preparation, which varies by manufacturer and biopharmaceutical product. Various methods have been used to fractionate or deplete mAbs from peptides. Natural digestion and molecular weight cutoff filtration have recently been used as alternative methods. However, these techniques are not standardized and may only apply to specific subclasses of mAbs, making them less suitable for quantifying problematic HCPs. For example, previous studies have shown that phospholipase B-like protein 2 (PLBL2) and cathepsin D interact with mAbs under protein A chromatography conditions and appear at higher levels in the eluent. These HCPs have the potential to induce immunogenicity or hydrolyze mAbs. Therefore, separating HCPs using protein A chromatography followed by LC-MS / MS analysis may not provide a complete picture of HCPs with specific risk levels for root cause investigation. Significant progress has been made in overcoming this problem by using pre-elution washing to disrupt HCP-mAb interactions and improve HCP removal via protein A chromatography. However, research reveals that the type of HCP co-eluted with the product varies from molecule to molecule, which poses a significant bottleneck in the biopharmaceutical industry, especially where short turnaround times are required.

[0009] Furthermore, the time-consuming sample preparation process required for HCP, including reduction, alkylation, desalting, and overnight digestion, can hinder high-throughput LC-MS analysis. Therefore, the lack of a rapid and universal method for HCP separation and digestion remains a major obstacle when time and sample throughput are critical limiting factors.

[0010] To address these challenges, this application provides an integrated strategy aimed at ensuring efficient and reliable sample preparation (isolation and digestion) of protein contaminants for different classes of recombinant proteins. This method aims to reduce the dynamic range of recombinant proteins relative to protein contaminants, providing a reproducible, straightforward, and easy-to-implement approach that can be performed in a standard laboratory setting. This method was used in a case study of mAb bioprocessing to investigate the identification and quantification of high-risk HCPs.

[0011] Recently, Fc receptor (FcγRIIIa) affinity chromatography has been used to improve the analytical characterization of mAbs by monitoring Fc glycan heterogeneity during process development and manufacturing [1, 2]. In this invention, an Fc receptor (FcγRIIIa) affinity column is used to deplete the mAb and separate the enriched HCP fraction. The fraction is then concentrated using a retardation filter, and the protein is precipitated with acetone. Subsequently, the protein precipitate is dissolved, denatured, reduced, and digested in a solution containing sodium deoxycholate (SDC) and N-dodecyl-β-D-maltose glycoside (DDM) as suitable detergents. Comparison of HCP fractions separated by FcγRIIIa and protein A chromatography shows a significant improvement in HCP identification when using FcγRIIIa. The newly developed method simplifies the sample preparation process, reduces variability, and shortens turnaround time. This advance has the potential to provide valuable insights into problematic or high-risk HCPs, thereby enhancing control strategies during purification evaluation in biopharmaceutical companies.

[0012] To reduce the variability in protein contaminant identification caused by different sample preparation procedures and low concentrations of protein contaminants, a more efficient sample preparation workflow is needed. This application discloses a novel procedure that combines protein contaminant separation on an Fc receptor affinity column with “single-tube” protein digestion. This method demonstrates a significant increase in the number of identified protein contaminants compared to protein A chromatography by reducing the ratio of recombinant protein to protein contaminants. This method is applicable to a wide range of recombinant proteins and provides a reproducible, direct, and easy-to-implement approach that can be performed in a standard laboratory. The disclosed sample preparation method will be valuable for monitoring protein contaminant levels in manufacturing processes and can be widely used for root cause investigations. Furthermore, this method can help assess the biosimilarity of candidates to reference products and guide process development to improve overall HCP clearance in downstream processes.

[0013] This application provides a rapid and efficient workflow for the separation and digestion of protein contaminants such as HCPs. Fc receptor (FcγRIIIa) affinity chromatography is used to separate HCP fractions through mAb depletion. HCPs in the HCP fractions can be precipitated and digested using a “single-tube” method, which improves digestion performance and prevents sample loss of problematic low-abundance protein contaminants. The method of this invention shows superiority over protein A affinity chromatography in monitoring problematic HCPs. Summary of the Invention

[0014] In one aspect, the present invention relates to a method for separating and analyzing one or more protein contaminants in a sample containing recombinant proteins and one or more protein contaminants, the method comprising: (a) fractionation of the sample containing recombinant proteins and one or more protein contaminants, wherein fractionation comprises contacting the sample with an Fc receptor affinity resin and collecting flow-through (FT) to obtain a fraction containing one or more protein contaminants; and (b) analysis of one or more protein contaminants in the fraction containing one or more protein contaminants. Attached Figure Description

[0015] Figure 1 Experimental workflow: The mAb is depleted using an Fc receptor column (e.g., FcγRIIIa), and the collected HCP fraction is concentrated (e.g., through a 10 kDa cutoff filter). The protein is precipitated (e.g., by adding acetone) and dissolved (e.g., in SDC / DDM solution), then reduced and digested (e.g., by trypsin). The peptide is analyzed by LC-MS / MS, and the raw file is searched against a CHO protein sequence database to identify the HCP.

[0016] Figure 2 FcγRIIIa affinity UV chromatograms of (A) mAb1 and (B) mAb2 after N-glycan removal with PNGase F (dashed trace) at 280 nm.

[0017] Figure 3 Analysis of mAb2 fragments digested with FabALACTICA. (A) FcγRIIIa affinity chromatogram at 280 nm using UV detection (FT window of the FcγRIIIa affinity column is approximately 5 to approximately 12 min); (B) RP-LC-MS analysis of the FT peak (fraction 1) (RT window of the RP-LC column is 5 to 20 min) and (C) peak at RT of 30–45 min (fraction 2); (D) deconvolution mass spectra of fraction 1 and (E) fraction 2.

[0018] Figure 4Analysis of mAb2 fragments digested with IdeS. (A) Chromatogram of FcγRIIIa at 280 nm using UV detection (FT window of the FcγRIIIa affinity column is approximately 5 to approximately 12 min); (B) RP-LC-MS analysis of the FT fraction (RT window of the RP-LC column is 5 to approximately 20 min); deconvolution mass spectra of the peaks at RT of (C) 14.8 min and (D) 16.8 min.

[0019] Figure 5 UPS1 protein identification and peptide intensity of all detected peptides after digestion at 37°C for 15 to 60 minutes. The values ​​above the bars represent the average sequence coverage of all identified UPS1 proteins. The sequence coverage levels of identified UPS1 proteins are represented by different colors.

[0020] Figure 6 Superimposed UV chromatograms (at 280 nm) of mAb1 obtained by FcγRIIIa affinity chromatography from three consecutive injections. Fraction 1 was collected and used for HCP identification in a bottom-up approach.

[0021] Figure 7 (A) Signal intensity (MS signal) distribution of all detected peptides of protein PLBL2 in each fraction of affinity chromatography for protein A and (B) FcγRIIIa.

[0022] Figure 8 : A schematic diagram of the protein A workflow described in [3]. In short, the workflow includes the following steps: (I) Equilibrate the protein A column with 10 mL of 20 mM Na2HPO4 pH 7.3 (HiTrap MabSelect SuRe, 1 mL, Sigma-Aldrich). (II) Dilute 5 mg mAb1 to a final volume of 2 mL (5 mg / mL concentration) with 20 mM Na2HPO4 pH 7.3 and load it onto the protein A column, and collect the flow-through (F1-protein A). (III) Load the pre-elution solution onto the column and collect the fraction (F2-protein A). (IV) Reequilibrate the column with 20 mM Na2HPO4 pH 7.3 (waste liquid). (V) Elute mAb1 with 5.0 mL of 100 mM glycine buffer pH 2.8 and collect the fraction (F3-protein A). Detailed Implementation

[0023] List of abbreviations used in this application Fractionation of samples containing recombinant proteins and one or more protein contaminants is used to deplete the recombinant proteins in the sample and thereby achieve relative enrichment of one or more protein contaminants to support efficient and sensitive detection and analysis, such as by mass spectrometry.

[0024] The contact of the sample with the Fc receptor affinity resin described herein produces two fractions: a binding fraction and a flow-through fraction. The binding fraction, also known as the retention fraction, contains all proteins in the sample that interact with the affinity resin used, i.e., proteins retained by the Fc receptor affinity resin and therefore eluted from the resin significantly later than the flow-through fraction. The flow-through fraction contains all proteins in the sample that do not interact with the affinity resin used, or substantially do not interact with it. As referred to herein, “collecting flow-through” means collecting the eluent or at least a portion of the eluent that flows out of the affinity resin under the elution conditions where the recombinant protein binds to the Fc receptor affinity resin. Therefore, “collecting flow-through” can refer to collecting the eluent or at least a portion of the eluent that flows out of the column under the elution conditions where the recombinant protein binds to the affinity resin contained in the column. This collected eluent can also be referred to as the collected flow-through. It contains at least a portion, preferably all, of proteins that are not bound to the Fc receptor affinity resin (not retained by the Fc receptor affinity resin). For ease of reading, the term “flow-through” is used throughout the specification, but it should be understood that it covers not only the flow-through of the column, but also any solution obtained after contacting the sample with the Fc receptor affinity resin that contains at least some, preferably all, proteins that are not bound to the Fc receptor affinity resin.

[0025] In a preferred embodiment, one or more protein contaminants are one or more host cell proteins. Host cell proteins are protein contaminants derived from host cells used to produce recombinant proteins. Recombinant proteins are typically produced in cell cultures using CHO, NSO, Sp2 / O, HEK293, or PER.C6 cells. Therefore, in a specific embodiment, one or more protein contaminants are host cell proteins derived from cell lines selected from the group consisting of CHO, NSO, Sp2 / O, HEK293, and PER.C6 cells. In a preferred embodiment, one or more protein contaminants are host cell proteins derived from CHO cell lines.

[0026] The method according to the invention includes the fractionation of a sample comprising recombinant proteins and one or more protein contaminants, wherein the fractionation comprises contacting the sample with an Fc receptor affinity resin and collecting the flowthrough to obtain a fraction comprising one or more protein contaminants. In some embodiments, the Fc receptor affinity resin comprises immobilized Fc receptors that bind to the recombinant proteins. This means that Fc receptors immobilized on the Fc receptor affinity resin are selected such that the Fc receptors bind to the recombinant proteins. In the sense of this application, the Fc receptors “bind” to the recombinant proteins when a specific interaction occurs between the Fc receptors and the recombinant proteins. This results in the recombinant proteins associating with the Fc receptor affinity resin, and a reduction in the amount of recombinant proteins in the flowthrough compared to the amount of recombinant proteins in the initial sample (before fractionation).

[0027] In one embodiment, an Fc acceptor affinity resin is a resin to which an Fc acceptor is preferably covalently bonded. The Fc acceptor is immobilized on the resin. The resin can be any known resin that is stable under conditions conventionally used for protein affinity purification (e.g., affinity chromatography), with polymethacrylate being a widely used example. In a preferred embodiment, the resin is used in the form of non-porous or porous polymethacrylate-based beads. Typical bead sizes are about 2 to 15 micrometers, for example, 5 or 10 micrometers. In the case of porous resins, typical nominal pore sizes can be about 100 nm. Thus, an Fc acceptor affinity resin is an Fc acceptor immobilized on a stationary phase resin and is typically used in the form of a chromatographic column packed with a resin containing an immobilized Fc acceptor.

[0028] In one embodiment, the Fc receptor affinity resin is an Fcγ receptor affinity resin or a neonatal Fc receptor (FcRn) affinity resin, preferably an Fcγ receptor affinity resin. The Fcγ receptor affinity resin can be a type I Fcγ receptor (FcγR) affinity resin, a type II Fcγ receptor (FcγR) affinity resin, or a type III Fcγ receptor (FcγR) affinity resin; preferably, the Fcγ receptor affinity resin is a type I Fcγ receptor (FcγR) affinity resin; more preferably, the type I Fcγ receptor (FcγR) affinity resin is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb affinity resins; even more preferably, the type I Fcγ receptor (FcγR) affinity resin is an FcγRIIIa affinity resin. Therefore, in a preferred embodiment, the Fc receptor affinity resin is a type I Fcγ receptor (FcγR) affinity resin. In a further preferred embodiment, the type I Fcγ receptor (FcγR) affinity resin is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb affinity resins. In a more preferred embodiment, the type I Fcγ receptor (FcγR) affinity resin is an FcγRIIIa affinity resin.

[0029] Fc acceptor affinity resins can be used in the form of chromatographic columns. Fc acceptor affinity resins in column form are used for column chromatography. Therefore, in one embodiment, fractionation involves contacting the sample with the Fc acceptor affinity resin, wherein the contact includes column chromatography.

[0030] Affinity chromatography typically involves the use of a stationary phase (e.g., an Fc receptor affinity resin) and a mobile phase. In the context of this invention, the mobile phase used for Fc receptor affinity chromatography can be any mobile phase suitable for use with the stationary phase used (i.e., the Fc receptor affinity column used). Chakrabarti et al.

[12] disclosed suitable conditions for protein purification using Fc receptor affinity chromatography.

[0031] In some embodiments, column chromatography using the Fc receptor affinity resin as described herein includes using a mobile phase that is an aqueous buffer having a pH between about 3 and about 8, preferably between about 4 and about 8, and more preferably between about 4.5 and about 7.5. In some embodiments, column chromatography includes using a mobile phase that is a citrate buffer (e.g., ammonium citrate) or an acetate buffer (e.g., ammonium acetate). In some embodiments, column chromatography includes using a mobile phase that is an aqueous buffer having a buffer concentration between about 10 mM and about 200 mM, preferably between about 20 mM and about 150 mM, and more preferably between about 30 mM and about 100 mM. In one specific embodiment, the buffer concentration is about 50 mM.

[0032] In some embodiments, column chromatography includes the use of a mobile phase that is a gradient of two aqueous buffer solutions. Typically, elution from the Fc acceptor affinity resin is achieved by gradually decreasing the pH of the mobile phase, for example by using a pH gradient, such as from about pH 7 to about pH 4, preferably from about pH 7.0 to about pH 4.0. Therefore, in some embodiments, the mobile phase is a gradient of two aqueous buffer solutions, wherein the first buffer solution has a pH between about 6 and about 7, preferably between about 6.0 and about 7.0, and the second buffer solution has a pH between about 3.5 and about 5, preferably between about 4 and about 5, more preferably between about 4.0 and about 5.0. In a preferred embodiment, the mobile phase is a gradient of two aqueous buffer solutions, wherein the first buffer solution has a pH of about 6.5 and the second buffer solution has a pH of about 4.5. In a specific embodiment, the mobile phase is a gradient of two aqueous citrate buffers having a buffer concentration between about 10 mM and about 200 mM, wherein the first buffer has a pH between about 6 and about 7, preferably between about 6.0 and about 7.0, and the second buffer has a pH between about 3.5 and about 5, preferably between about 4 and about 5, more preferably between about 4.0 and about 5.0.

[0033] As used herein, the term "recombinant protein" refers to a protein produced through recombinant synthesis, preferably from a cell line. Recombinant proteins can be used for therapeutic purposes. Therefore, recombinant proteins can be therapeutic proteins. However, the availability of the disclosed methods is not limited to recombinant proteins with therapeutic activity, but is applicable to any recombinantly produced protein.

[0034] The method according to the invention relies on the retention of recombinant proteins by Fc receptor affinity resins, while one or more protein contaminants are not retained by the Fc receptor affinity resins. Therefore, in some embodiments, the recombinant protein binds to the Fc receptor affinity resin. In the sense of this application, the recombinant protein “binds” to the Fc receptor affinity resin when a specific interaction occurs between the Fc receptor and the recombinant protein. This results in the recombinant protein associating with the Fc receptor affinity resin, and a reduction in the amount of recombinant protein in the flowthrough compared to the amount of recombinant protein in a sample (before fractionation) containing both the recombinant protein and one or more protein contaminants.

[0035] In some embodiments, the recombinant protein is an antibody or its antigen-binding fragment, or an Fc fusion protein. In a preferred embodiment, the recombinant protein is an antibody or its antigen-binding fragment. An Fc fusion protein is a protein containing an Fc domain that has been recombinantly linked to a peptide or protein of interest. The Fc domain of an Fc fusion protein may, for example, be derived from an IgG-type constant region as described herein.

[0036] Therefore, in one embodiment, the Fc domain is an IgG-type Fc domain. In a preferred embodiment, the Fc domain is an IgG1, IgG2, IgG3, or IgG4-type Fc domain. In a more preferred embodiment, the Fc domain is an IgG1, IgG2, or IgG4-type Fc domain. In an even more preferred embodiment, the Fc domain is an IgG1 or IgG4-type Fc domain.

[0037] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody or its antigen-binding fragment.

[0038] As used herein, the term “monoclonal antibody” refers to an antibody derived from a substantially homogeneous population of antibodies, i.e., individual antibodies comprising that population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) present in small amounts.

[0039] The term "human antibody" includes antibodies having variable and constant regions that substantially correspond to sequences of human immunoglobulins known in the art, including, for example, those derived from Kabat. et al. [6] The antibodies described herein. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., via...). in vitroRandom or site-specific mutagenesis or through in vivo Mutations introduced by somatic mutations, such as in CDR, and especially CDR3. Human antibodies may have at least one, two, three, four, five or more sites where amino acid residues not encoded by human immunoglobulin sequences are substituted.

[0040] In addition, the term “antibody” as used herein also refers to derivatives or variants of the antibodies described herein that exhibit the same specificity as the antibodies described herein. Examples of antibody variants include humanized variants of nonhuman antibodies, affinity-matured antibodies (see, for example, Hawkins et al. [7] and Lowman et al. [8]), and antibody mutants with altered effector functions (see, for example, Kontermann and Dübel [9]).

[0041] In addition to full-length antibodies, the term “antibody” as used herein also includes antibody fragments and antibody derivatives, such as scFv-Fc, hcIgG, or IgNAR antibodies. Further envisioned are (bispecific) single-chain antibodies, “FcDART” antibodies and “IgGDART” antibodies, Triomab antibodies, CrossMab antibodies, otho-Fab IgG antibodies, and DVD-IgG (Kontermann 2015

[10] ).

[0042] In some embodiments, the recombinant protein comprises an Fc domain or a fragment of an Fc domain, preferably a glycosylated Fc domain or a fragment of a glycosylated Fc domain. In some embodiments, the recombinant protein comprises an Fc domain or a fragment of an Fc domain and is an antibody or its antigen-binding fragment or an Fc fusion protein.

[0043] As used herein, the term "Fc domain" refers to a polypeptide that contains the constant region of an antibody (excluding one or more variable domains and the first constant region, the immunoglobulin domain). For IgG antibodies, the Fc domain contains the immunoglobulin domains CH2 and CH3, as well as a hinge region located between the immunoglobulin domains CH2 and CH1.

[0044] In some embodiments, the Fc domain is a mouse Fc domain or a human Fc domain. In a preferred embodiment, the Fc domain is a human Fc domain. In some embodiments, the Fc domain includes immunoglobulin domains CH2 and CH3 and a hinge region located between immunoglobulin domains CH2 and CH1, wherein the CH2 domain, CH3 domain, and hinge region are human-derived.

[0045] In a further embodiment, the recombinant protein comprises an IgG-type constant region. In some embodiments, the recombinant protein comprises an IgG-type constant region and is an antibody or its antigen-binding fragment or Fc fusion protein.

[0046] As used herein, the IgG type constant region comprises immunoglobulin domains CH1, CH2, and CH3, together with a hinge region located between immunoglobulin domains CH2 and CH1. The IgG type constant region can be an IgG1, IgG2, IgG3, or IgG4 type constant region. Preferably, the IgG type constant region is an IgG1, IgG2, or IgG4 type constant region. More preferably, the IgG type constant region is an IgG1 or IgG4 type constant region.

[0047] In some embodiments, the IgG type constant region is a mouse IgG type constant region or a human IgG type constant region. In a preferred embodiment, the IgG type constant region is a human IgG type constant region.

[0048] Therefore, in some embodiments, the recombinant protein is an IgG antibody, preferably an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, more preferably an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody, and even more preferably an IgG1 antibody or an IgG4 antibody.

[0049] The interaction between the Fc receptor and its substrate can depend on the glycosylation profile of the substrate [1, 2, 12]. Therefore, the level of retention achievable with Fc receptor-based resins (e.g., Fc receptor-based columns) can depend on whether the recombinant protein substrate is glycosylated, particularly N-glycosylated. This is especially true for embodiments where the recombinant protein is an antibody or its antigen-binding fragment or an Fc fusion protein, or where the recombinant protein contains an Fc domain or an IgG-type constant region. For such recombinant proteins glycosylated within the Fc domain or within the IgG-type constant region, retention on Fc receptor-based resins is enhanced. As described in [1, 2], larger N-glycans generally result in higher affinity of the Fc receptor for the recombinant protein substrate, and thus longer retention times on Fc receptor affinity resins (e.g., FcγRIIIa affinity resins). The number of terminal galactose sugars within the N-glycan has also been shown to be positively correlated with retention time on Fc receptor affinity resins (e.g., FcγRIIIa affinity resins).

[0050] Therefore, in some embodiments, the recombinant protein (e.g., an antibody or its antigen-binding fragment or Fc fusion protein) is glycosylated. In a preferred embodiment, the recombinant protein (e.g., an antibody or its antigen-binding fragment or Fc fusion protein) comprises an N-glycan, i.e., is N-glycosylated. In a further preferred embodiment, the recombinant protein comprises an N-glycan at position 297 or the corresponding amino acid according to EU number. In a particularly preferred embodiment, the recombinant protein is an antibody or its antigen-binding fragment (e.g., an IgG-type antibody) or an Fc fusion protein, or comprises an Fc domain or an IgG-type constant region, and comprises an N-glycan at position 297 (according to Edelman et al.

[11] ) or the corresponding amino acid according to EU number.

[0051] The glycan linked to asparagine residues is referred to herein as an N-glycan. In the context of this invention, the structure of the N-glycan and the number of sugar residues contained therein may affect the retention of recombinant proteins by Fc receptor affinity resins (an exemplary overview in this regard is provided in

[13] ). For example, the N-glycan may contain a core fucose sugar or may not contain a core fucose sugar. The N-glycan may also be an oligosaccharide having 3 to 20, preferably 3 to 15, particularly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 sugar residues.

[0052] In one specific embodiment, the recombinant protein comprises an Fc domain (e.g., an IgG-type Fc domain) and the Fc domain is glycosylated. In a preferred embodiment, the recombinant protein comprises an Fc domain (e.g., an IgG-type Fc domain) and the Fc domain comprises an N-glycan (i.e., is N-glycosylated). In another preferred embodiment, the recombinant protein comprises an Fc domain (e.g., an IgG-type Fc domain) and the Fc domain contains an N-glycan at position 297 according to EU number. In some embodiments, the N-glycan contained in the Fc domain is an oligosaccharide having 3 to 20, preferably 3 to 15, particularly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 sugar residues.

[0053] In a further specific embodiment, the recombinant protein comprises an IgG-type constant region, and the IgG-type constant region is glycosylated. In a preferred embodiment, the recombinant protein comprises an IgG-type constant region, and the IgG-type constant region comprises an N-glycan. In a preferred embodiment, the recombinant protein comprises an IgG-type constant region, and the IgG-type constant region comprises an N-glycan at position 297 according to EU number. In some embodiments, the N-glycan within the IgG-type constant region is an oligosaccharide having 3 to 20, preferably 3 to 15, particularly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 sugar residues.

[0054] Given that antibody-dependent cell-mediated cytotoxicity (ADCC activity) requires an interaction between an antibody (or its antigen-binding fragment) and an Fc receptor, in some embodiments, recombinant proteins (e.g., antibodies or their antigen-binding fragments) possess ADCC activity. ADCC activity can be measured, for example, as described by Kiyoshi et al. [1].

[0055] Fractions containing one or more protein contaminants obtained after fractionation of a sample containing recombinant proteins may be subject to concentration of one or more protein contaminants. Therefore, in one embodiment, the analysis in step b) includes subjecting the fraction containing one or more protein contaminants to concentration of one or more protein contaminants. In one embodiment, concentration of one or more protein contaminants includes (i.) precipitation of one or more protein contaminants; and (ii.) dissolution of the precipitated one or more protein contaminants in a suitable solvent. In some embodiments, concentration of one or more protein contaminants further includes reducing the volume of the fraction containing one or more protein contaminants prior to precipitation of the one or more protein contaminants.

[0056] Therefore, in one specific embodiment, the method includes subjecting a fraction containing one or more protein contaminants to concentration of one or more protein contaminants, wherein the concentration of one or more protein contaminants includes precipitation of one or more protein contaminants; and dissolving the precipitated one or more protein contaminants in a suitable solvent.

[0057] In a further specific embodiment, the method includes subjecting a fraction containing one or more protein contaminants to concentration of one or more protein contaminants, wherein the concentration of one or more protein contaminants includes a reduction in volume of the fraction containing one or more protein contaminants, followed by precipitation of one or more protein contaminants; and dissolution of the precipitated one or more protein contaminants in a suitable solvent.

[0058] Volume reduction can be achieved by various suitable methods known to those skilled in the art. In one embodiment, volume reduction of a fraction containing one or more protein contaminants includes the use of a molecular weight cutoff (MWCO) filter. In a specific embodiment, the MWCO filter has a molecular weight cutoff value between about 2 kDa and about 30 kDa, between about 3 kDa and about 25 kDa, between about 4 kDa and about 20 kDa, or between about 5 kDa and about 15 kDa. In a preferred embodiment, the MWCO filter has a molecular weight cutoff value between about 5 kDa and about 15 kDa. In a further embodiment, the MWCO filter has a molecular weight cutoff value of about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, or about 15 kDa. In a preferred embodiment, the MWCO filter has a molecular weight cutoff value of about 10 kDa.

[0059] Precipitation of one or more protein contaminants is typically followed by sedimentation of the precipitated protein contaminants (e.g., by centrifugation), removal of the supernatant, and disposal of the supernatant. The resulting protein precipitate contains the analyte of interest, i.e., one or more protein contaminants, and is used for further processing and analysis as described herein. Therefore, in some embodiments, precipitation of one or more protein contaminants is followed by sedimentation of the precipitated protein contaminants and removal of the supernatant. After sedimentation, the resulting protein precipitate may be dried. Drying can be performed by common methods known to those skilled in the art, such as air drying, lyophilization, or vacuum centrifugation.

[0060] Protein precipitation can be achieved by various methods known to those skilled in the art. In some embodiments, precipitation of one or more protein contaminants includes the addition of a precipitant. Preferably, the precipitant is selected from the group consisting of trichloroacetic acid (TCA), acetone, ammonium sulfate, chloroform, methanol, and water, as well as the group consisting of chloroform and methanol. In a further preferred embodiment, the precipitant is acetone. The precipitant may be cooled, i.e., it may have a temperature between about 2°C and about 8°C, for example, at about 4°C.

[0061] The reagent (precipitant) added for the purpose of precipitating one or more protein contaminants is typically added in an amount sufficient to cause complete or substantially complete precipitation of the proteins contained in a fraction containing one or more protein contaminants. In some embodiments, precipitation of one or more protein contaminants comprises adding a precipitant in an amount at least about 2:1, at least about 3:1, at least about 4:1, or at least about 5:1 relative to the volume of the fraction containing one or more protein contaminants (optionally after the volume reduction of the fraction and / or the addition of a saline solution). In a preferred embodiment, precipitation of one or more protein contaminants comprises adding a precipitant in an amount at least about 2:1 relative to the volume of the fraction containing one or more protein contaminants (optionally after the volume reduction of the fraction and / or the addition of a saline solution). In a further preferred embodiment, precipitation of one or more protein contaminants comprises adding a precipitant in an amount at least about 3:1 relative to the volume of the fraction containing one or more protein contaminants (optionally after the volume reduction of the fraction and / or the addition of a saline solution) (e.g., adding at least 300 µL of reagent to 100 µL of a fraction containing one or more protein contaminants). In a more preferred embodiment, precipitation of one or more protein contaminants includes adding a precipitant in an amount of at least about 4:1 relative to the volume of the fraction containing one or more protein contaminants.

[0062] Precipitation of one or more protein contaminants may include the addition of a brine solution. Preferably, the brine solution is added prior to the addition of a precipitant. More preferably, the brine solution is an aqueous solution of a salt selected from the group consisting of NaCl. In a preferred embodiment, the brine solution is an aqueous solution of NaCl. The salt contained in the brine solution is not a precipitant as disclosed herein, for example, it is not ammonium sulfate.

[0063] In some embodiments, the saline solution contains the salt at a concentration between about 50 mM and about 500 mM, about 100 mM and about 400 mM, or about 150 mM and about 300 mM. In a preferred embodiment, the saline solution contains the salt at a concentration between about 150 mM and about 300 mM. In further embodiments, the saline solution contains the salt at a concentration of about 100 mM, about 200 mM, about 300 mM, about 400 mM, or about 500 mM. In a preferred embodiment, the saline solution contains the salt at a concentration of about 200 mM.

[0064] After the addition of reagents intended for the precipitation of one or more protein contaminants, the resulting suspension may be maintained at a reduced temperature to promote protein precipitation prior to further processing (e.g., before the precipitated one or more protein contaminants settle). In some embodiments, the suspension is maintained at a temperature of about -20°C or lower for at least about 5 minutes. In some embodiments, the suspension is maintained at a temperature of about -60°C or lower for at least about 8 minutes. In some embodiments, the sample is maintained at a temperature of about -65°C or lower for at least about 10 minutes.

[0065] The sedimentation of one or more protein contaminants is typically achieved by centrifugation. In some embodiments, sedimentation of one or more protein contaminants comprises centrifuging at a radial force of at least about 10,000 g for at least about 5 minutes. In a further embodiment, sedimentation of one or more protein contaminants comprises centrifuging at a radial force of at least about 14,000 g for at least about 10 minutes. Centrifugation may be performed at a reduced temperature (relative to room temperature), for example, between about 2°C and about 8°C, for example, at about 4°C.

[0066] Therefore, in some embodiments, the method according to the invention includes (a) Fractionation of a sample containing recombinant proteins and one or more protein contaminants, wherein the fractionation comprises contacting the sample with an Fc receptor affinity resin and collecting the flow-through to obtain a fraction containing one or more protein contaminants. (b) Analysis of one or more protein contaminants in a fraction containing one or more protein contaminants, wherein the analysis includes A fraction containing one or more protein contaminants is subjected to concentration of one or more protein contaminants, wherein the concentration of one or more protein contaminants includes a reduction in volume of the fraction containing one or more protein contaminants, followed by the addition of a brine solution, followed by precipitation of one or more protein contaminants, including the addition of a precipitant selected from the group consisting of trichloroacetic acid (TCA), acetone, ammonium sulfate, chloroform and a combination of methanol and water, and a combination of chloroform and methanol, followed by maintaining the resulting suspension at a reduced temperature, followed by sedimentation of the precipitated one or more protein contaminants (e.g., by centrifugation), removal of the supernatant and disposal of the supernatant; and dissolution of the precipitated one or more protein contaminants in a suitable solvent.

[0067] One or more protein contaminants that have been precipitated are dissolved in a suitable solvent. The suitable solvent and possible additives should generally be compatible with the subsequent chemical reduction and protein hydrolysis digestion; that is, they should not interfere with any of these reactions.

[0068] A suitable solvent is preferably a suitable aqueous buffer solution. Suitable aqueous buffer solutions are typically based on ammonium salts. Buffer salts can be selected from the group consisting of ammonium formate, tris-HCl, ammonium acetate, and ammonium bicarbonate or ammonium carbonate. In a preferred embodiment, the aqueous buffer solution is an aqueous solution of ammonium bicarbonate. The concentration of the aqueous buffer solution can be between about 10 mM and about 100 mM, between about 20 mM and about 90 mM, between about 30 mM and about 70 mM, and between about 40 mM and about 60 mM. In a specific embodiment, the concentration of the aqueous buffer solution is about 50 mM. In some embodiments, the pH of the aqueous buffer solution is between about 7 and about 9, preferably between about 7.0 and about 9.0. In a preferred embodiment, the pH of the aqueous buffer solution is between about 7.4 and about 8.6. In a more preferred embodiment, the pH of the aqueous buffer solution is between about 7.6 and about 8.4. In a particularly preferred embodiment, the pH of the aqueous buffer solution is about 8, more preferably about 8.0.

[0069] Therefore, in a preferred embodiment, the aqueous buffer is an aqueous solution of ammonium bicarbonate, wherein the buffer concentration is between about 10 mM and about 100 mM, and the pH of the aqueous buffer is between about 7 and about 9, preferably between about 7.0 and about 9.0. In a particularly preferred embodiment, the aqueous buffer is an aqueous solution of ammonium bicarbonate, wherein the buffer concentration is about 50 mM, and the pH of the aqueous buffer is about 8, preferably about 8.0.

[0070] A suitable solvent may be an aqueous buffer solution containing a detergent. At least one detergent is preferably one that does not interfere with subsequent analysis by liquid chromatography-mass spectrometry. At least one detergent may be N-octyl-β-glucopyranoside, sodium deoxycholate, N-dodecyl-β-D-maltose, or a combination thereof. In some embodiments, a suitable solvent is an aqueous buffer solution containing a detergent, preferably selected from the group consisting of sodium deoxycholate, N-dodecyl-β-D-maltose, and combinations thereof. In a particularly preferred embodiment, the detergent is a combination of sodium deoxycholate and N-dodecyl-β-D-maltose.

[0071] The detergent is preferably used at a concentration that ensures it does not interfere with subsequent analyses performed by liquid chromatography-mass spectrometry. In some embodiments, the aqueous buffer contains a detergent at a concentration between about 0.05% and about 1.0% (w / v). Sodium deoxycholate (SDC) can be used at a concentration ranging from about 0.05% to about 0.8% (w / v). N-Dodecyl-β-D-maltodextrin can be used at a concentration ranging from about 0.01% to about 0.5% (w / v). Therefore, in a specific embodiment, the aqueous buffer contains sodium deoxycholate at a concentration between about 0.05% and about 0.8% (w / v) and N-dodecyl-β-D-maltodextrin at a concentration between about 0.01% and about 0.5% (w / v). In a preferred embodiment, the aqueous buffer comprises sodium deoxycholate at a concentration of about 0.1% to about 0.6% (w / v) and N-dodecyl-β-D-maltodextrin at a concentration of about 0.04% to about 0.5% (w / v). In a more preferred embodiment, the aqueous buffer comprises sodium deoxycholate at a concentration of about 0.2% to about 0.4% (w / v) and N-dodecyl-β-D-maltodextrin at a concentration of about 0.05% to 0.5%, preferably about 0.06% to about 0.2% (w / v).

[0072] In some embodiments, the analysis in step b) includes the fragmentation of one or more protein contaminants. In a preferred embodiment, the fragmentation of one or more protein contaminants is performed by protein hydrolysis digestion. In some embodiments, the method includes the chemical reduction of one or more protein contaminants before or after fragmentation, preferably before fragmentation.

[0073] Therefore, in some embodiments, the method includes the chemical reduction of one or more protein contaminants, followed by the fragmentation of one or more protein contaminants by protein hydrolysis digestion.

[0074] In a specific embodiment, the method includes (a) fractionation of a sample containing recombinant proteins and one or more protein contaminants, wherein fractionation includes contacting the sample with an Fc receptor affinity resin and collecting the flow-through to obtain a fraction containing one or more protein contaminants; (b) analysis of one or more protein contaminants in the fraction containing one or more protein contaminants, wherein the analysis includes subjecting the fraction containing one or more protein contaminants to concentration of one or more protein contaminants, wherein concentration of one or more protein contaminants includes precipitation of one or more protein contaminants, dissolution of the precipitated one or more protein contaminants in a suitable solvent, chemical reduction of one or more protein contaminants, followed by fragmentation of one or more protein contaminants by protein hydrolysis digestion.

[0075] Any commonly used reagents for chemical reduction prior to mass spectrometry analysis can be used in the method of this invention. In some embodiments, chemical reduction includes using a reducing agent selected from the group consisting of dithiothreitol (DTT), tris(3-hydroxypropyl)phosphine (THPP), and tris(2-carboxyethyl)phosphine (TCEP). In a preferred embodiment, chemical reduction includes using dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In a particularly preferred embodiment, chemical reduction includes using tris(2-carboxyethyl)phosphine (TCEP).

[0076] The reducing agent is used at a concentration that results in quantification or substantially quantification of the peptides in the sample. In some embodiments, the reducing agent is used at a final concentration between about 1 mM and about 10 mM, between about 2 mM and about 9 mM, between about 3 mM and about 7 mM, or between about 4 mM and about 6 mM. In a preferred embodiment, the reducing agent is used at a final concentration between about 3 mM and about 7 mM. In a more preferred embodiment, the reducing agent is used at a final concentration between about 4 mM and about 6 mM.

[0077] Chemical reduction of proteins or peptides for subsequent mass spectrometry analysis is typically performed at elevated temperatures. Therefore, in some embodiments, the chemical reduction is performed between about room temperature and about 60°C, preferably between about 40°C and about 60°C. In a more preferred embodiment, the chemical reduction is performed between about 45°C and about 55°C. In an even more preferred embodiment, the chemical reduction is performed at about 50°C.

[0078] Proteolytic digestion for subsequent mass spectrometry analysis is typically performed using proteases that produce regular cleavage patterns. In some embodiments, proteolytic digestion includes using a protease selected from the group consisting of trypsin, chymotrypsin, ArgC, GluC, AspN, LysC, LysN, elastase, and proteinase K, and combinations thereof. In some embodiments, proteolytic digestion involves using a specific protease (such as trypsin). In some embodiments, proteolytic digestion involves using a combination of two specific proteases (such as trypsin and LysC). In a preferred embodiment, proteolytic digestion includes using trypsin and LysC. In a further preferred embodiment, proteolytic digestion includes using trypsin.

[0079] The proteases used for protein hydrolysis prior to mass spectrometry analysis are employed at their respective optimal temperatures (e.g., about 37°C). Therefore, in some embodiments, protein hydrolysis is performed at temperatures between about 30°C and about 60°C, preferably between about 34°C and about 40°C, more preferably between about 35°C and about 39°C, and even more preferably between about 36°C and about 38°C. In a particularly preferred embodiment, protein hydrolysis is performed at about 37°C.

[0080] Proteases can be used in varying amounts. In some embodiments, protein hydrolysis includes using one or more proteases at a ratio (w / w, protease to protein substrate) between about 1:5 and about 1:100, preferably between about 1:5 and about 1:40, more preferably between about 1:10 and about 1:30, and even more preferably between about 1:15 and about 1:25. In a particularly preferred embodiment, protein hydrolysis includes using one or more proteases at a ratio (w / w, protease to protein substrate) of about 1:20.

[0081] In some embodiments, proteolytic digestion is allowed to proceed for at least about 15 minutes. In a further embodiment, proteolytic digestion is allowed to proceed for at least about 30 minutes. In even further embodiments, proteolytic digestion is allowed to proceed for at least about 45 minutes. In even further embodiments, proteolytic digestion is allowed to proceed for at least about 60 minutes. Regarding the duration, proteolytic digestion may be allowed to proceed overnight. In a preferred embodiment, proteolytic digestion is allowed to proceed for about 15 minutes to about 90 minutes. In a more preferred embodiment, proteolytic digestion is allowed to proceed for about 30 minutes to about 60 minutes.

[0082] The method may further include the identification and / or quantification of one or more individual protein contaminants in a fraction containing one or more protein contaminants.

[0083] In one embodiment, the analysis of one or more protein contaminants in step b) includes the identification of one or more individual protein contaminants, preferably peptide analysis by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the liquid chromatography-mass spectrometry is liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0084] In some embodiments, the method according to the invention includes (a) fractionation of a sample comprising recombinant proteins and one or more protein contaminants, wherein fractionation comprises contacting the sample with an Fc receptor affinity resin and collecting the flow-through to obtain a fraction comprising one or more protein contaminants; (b) analysis of one or more protein contaminants in the fraction comprising one or more protein contaminants, wherein the analysis comprises subjecting the fraction comprising one or more protein contaminants to concentration of one or more protein contaminants, wherein concentration of one or more protein contaminants comprises precipitation of one or more protein contaminants, dissolution of the precipitated one or more protein contaminants in a suitable solvent; chemical reduction of one or more protein contaminants, followed by fragmentation of one or more protein contaminants by proteolytic digestion, and identification of one or more individual protein contaminants by peptide analysis by liquid chromatography-mass spectrometry (LC-MS).

[0085] As used herein, mass spectrometry (MS) is an analytical technique that filters, detects, identifies, and / or measures compounds by their mass-to-charge ratio, “Da / e” (also commonly represented by the symbol “m / z”). MS techniques typically involve (1) ionizing the compound and potentially fragmenting it; and (2) detecting the molecular weight of the charged compound and / or fragment ions and calculating the mass-to-charge ratio (m / z). Compounds can be ionized and detected by any suitable method. A mass spectrometer typically includes an ionizer and an ion detector.

[0086] Tandem mass spectrometry (MS / MS) is a mass spectrometry method in which precursor ions generated from a sample are selected by a first mass filter / analyzer and then transferred to a collision cell, where they are fragmented by collisions with neutral gas molecules, producing daughter (or "product") ions. The daughter ions are then subjected to mass analysis by a second mass filter / analyzer, and the resulting daughter ion spectra can be used to determine the structure and thereby identify the precursor (or "progenitor") ion. Tandem mass spectrometry is particularly useful for the analysis of complex mixtures such as biomolecules.

[0087] As used herein, liquid chromatography (LC) refers to the process of selectively retaining one or more components in a fluid solution when the fluid is uniformly permeated through a subdivided column or through capillary channels. Retention is caused by the distribution of components in a mixture between one or more stationary phases and the fluid as the bulk fluid (i.e., the mobile phase) moves relative to the stationary phase. Liquid chromatography includes, but is not limited to, reversed-phase liquid chromatography (RPLC), high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UHPLC), supercritical fluid chromatography (SFC), and ion chromatography. In some embodiments, the liquid chromatography performed as part of LC-MS analysis is reversed-phase (RP) liquid chromatography.

[0088] RP chromatography is based on a nonpolar stationary phase and a polar mobile phase. Stationary phases commonly used in RP reversed-phase (RP) liquid chromatography include silica gel resins functionalized with various nonpolar functional groups, such as alkyl functional groups. The alkyl-functionalized resin surface may include C-4, C-8, or C-18 functionalized resins, preferably C-18 functionalized resins.

[0089] Peptide analysis by liquid chromatography-mass spectrometry (LC-MS) can be performed using a variety of suitable instruments. Suitable LC-MS instruments are known to those skilled in the art and include, for example, the Orbitrap mass spectrometer from Thermo Fisher Scientific Inc.

[0090] Suitable solvent systems for reversed-phase liquid chromatography include a mobile phase composed of water and an organic solvent (e.g., methanol, ethanol, propanol, or acetonitrile) in varying ratios. The mobile phase may optionally contain a reagent for pH adjustment, such as acetic acid, formic acid, trifluoroacetate, ammonia, ammonium acetate, ammonium formate, or combinations thereof. In a preferred embodiment, the mobile phase comprises water, acetonitrile, and formic acid.

[0091] Following mass spectrometry analysis, peptides are mapped to peptide databases corresponding to the sources of one or more protein contaminants (e.g., cell lines used to produce recombinant proteins). Protein sequence data are publicly available, for example, from uniprot.org. For example, the CHO reference proteome is deposited with reference number UP000001075. Sequences of common contaminants can be added to the reference proteome used, such as keratin, trypsin (UniProt ID P00761), and recombinant protein sequences. The software digests the proteins in the database using the same protease (e.g., trypsin) used for the proteolytic digestion of the protein contaminants. Computer simulation Digestion. Calculate the mass of these peptides and compare them to a list of peaks with previously measured masses. If protein sequences in a reference database produce a large number of predicted masses that match the experimental mass, there is evidence that the protein is present in the original sample. Peptides can be fragmented using MS / MS to provide higher quality data and achieve more precise peptide identification. Perform statistical analysis on the results and use possible matches to identify and optionally quantify one or more protein contaminants in the sample.

[0092] Therefore, in some embodiments, peptide analysis and identification of individual protein contaminants by liquid chromatography-mass spectrometry (LC-MS) include comparing mass spectrometry data with data obtained by reference proteomics. Computer simulation A database of peptides generated by protein hydrolysis and digestion was compared.

[0093] It will be apparent to those skilled in the art that these embodiments and items merely depict instances of a variety of possibilities. Therefore, the embodiments shown herein should not be construed as limiting these features and configurations. Any possible combination and configuration of the described features can be selected according to the scope of the invention.

[0094] Example Materials and methods chemicals Tris(2-carboxyethyl)phosphine (TCEP), ammonium bicarbonate (ABC), ultrapure formic acid, acetic acid, guanidine hydrochloride, chloroform, methanol, acetonitrile (ACN), water, trifluoroacetic acid (TFA), sodium deoxycholate (SDC), and universal proteomics standards (UPS-1) were purchased from Sigma-Aldrich (St. Louis, MO). Vivaspin 500 (10 kDa MWCO) was purchased from Cytiva (Marlborough, MA). Sequencing-grade trypsin / Lys-C mixture and N-dodecyl-β-D-maltose glycoside (DDM) were purchased from Promega (Milwaukee, WI) and Thermo Fisher Scientific (Waltham, MA), respectively. IdeS and FabALACTICA were purchased from Genovis. PNGaseF was purchased from New England BioLabs.

[0095] Sample preparation for affinity chromatography Lonza internally used recombinant expression technology with CHO cell lines and standard purification procedures to produce mAb1 samples. mAb2 samples (8670 NIST mAb) were purchased from NIST. After incubation at 37°C for 3 hours, N-glycans were removed from the samples using PNGase F. Following the manufacturer's recommendations, the Fc and Fc / 2 fragments of mAb2 were prepared by digestion with FabALACTICA and IdeS, respectively.

[0096] Affinity chromatography Flow-through (FT) fractions were collected using an FcR-IIIA-5PW 7.8 mm x 7.5 cm column (P / N 0023532, TOSOH Bioscience, King of Prussia, PA). Mobile phase A was 50 mM citrate at pH 6.5, and mobile phase B was 50 mM citrate at pH 4.5. All samples were buffer-exchanged (3 times) with mobile phase A before injection. Samples were injected at a flow rate of 0.3 mL / min, the column temperature was 15 °C, and UV detection was performed at 280 nm.

[0097] To shorten HPLC run time, the gradient elution procedure was modified to a trap and elution method, as follows: After loading with 100% mobile phase A for 15 minutes, the gradient was switched to 100% B within 1 minute, and the flow rate was then maintained at 0.4 mL / min for 30 minutes. Before starting a new injection, the column was equilibrated with 100% mobile phase A for 27 minutes.

[0098] Protein A chromatography was performed using a HiTrap MabSelect SuRe column, following the procedure reported previously [3]. Figure 8 These steps are shown in more detail.

[0099] Sample preparation for peptide mapping analysis Protein precipitation with acetone Using a Vivaspin filter with a 10 kDa cutoff membrane and centrifuged at 10,000 g and 20 °C for approximately 20 min, the FT fraction collected during affinity chromatography (approximately 3.0 mL) was concentrated to approximately 100 μL of concentrate. Proteins in the concentrate were precipitated with acetone using a procedure similar to that described by Jiang et al. [4]. Briefly, the sample was diluted (1:1) with 200 mM NaCl, then 800 μL of cold acetone (4 °C) was added to the sample, and the solution was incubated in a refrigerator at ≤-65 °C for 10 min. After centrifugation (at 14,000 g and 4 °C) for 10 min, the liquid was carefully removed and discarded, and the remaining protein precipitate was used for proteomics sample preparation.

[0100] trypsin digestion The protein precipitate was dissolved in 15 μL of 50 mM ammonium bicarbonate solution containing SDC (2.3% w / v) and DDM (0.66% w / v). After vortex mixing at room temperature, the mixture was diluted by adding an additional 74 μL of 50 mM ammonium bicarbonate solution. The protein was reduced by adding 1 μL of 500 mM TCEP and incubating the solution at 50 °C for 10 min. The sample was digested with a trypsin / LysC mixture (1:20 enzyme to protein ratio; the trypsin:LysC ratio could be 1:1, w / w) at 37 °C for 1 h, and then directly transferred to HPLC vials for LC-MS / MS analysis as described below. The final concentrations of SDC and DDM before injection were 0.35% w / v and 0.1% w / v, respectively. The amount of protease required for digestion was calculated based on the assumption that the material loaded in the FcγRIIIa column contained 200 ppm HCP.

[0101] Liquid chromatography-mass spectrometry LC-MS / MS analysis was performed using a system consisting of a Vanquish Neo UHPLC instrument coupled with an Orbitrap Fusion Lumos mass spectrometer (ThermoFisher Scientific, Bremen, Germany). For peptide mapping analysis, NanoEase was used. m / z Peptides in digested samples were separated using a Peptide CSH C18 column (130 Å, 1.7 µm, 0.3 mm x 150 mm, Waters Corporation). The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile (B). Peptides were separated and eluted using a linear gradient from 1% B to 40% B over 75 minutes at a flow rate of 5 μL / min, followed by 98% B over 5 minutes. The column was then washed with 98% B for 3 minutes and conditioned with 1% B for 10 minutes before the next injection.

[0102] The mass spectrometer operates in data-dependent mode, with a range of 200-2000. m / z The spray voltage was 3500 kV, and the heating capillary temperature was 325℃. The full-scan spectrum was obtained at a resolution of 120,000 (at 400...). m / z (Full width at half maximum resolution), using 2.0 x 10 5 Automatic gain control (AGC) target values ​​were recorded with a maximum injection time of 100 ms. Up to 20 of the strongest ions with 2–8 charges were selected for high-energy c-trap dissociation (HCD) at 30% of the normalized collision energy. Fragment spectra were obtained at an isolation width of 2.5 Da and a resolution of 15,000, using a 5.0 x 10⁻⁶ pixel size. 4 The AGC target value and the maximum injection time of 200 ms were recorded. Dynamic exclusion was activated for 20 seconds within a 10 ppm window for precursor selection. Fragment ions were recorded on the Orbitrap analyzer.

[0103] Data Analysis For peptide mapping analysis, tolerances of 5 ppm and 20 ppm were applied to peptides identified in MS and MS / MS analyses, respectively, during database searches. A search was performed on the CHO protein sequence database obtained from UniProt (reference proteomics UP000001075, last modified January 24, 2019, downloaded October 10, 2022, containing 23,886 sequences), which included sequences of common contaminants such as keratin and trypsin (UniProt IDP00761) as well as the analyzed mAbs. The search included methionine and tryptophan oxidation and asparagine deamidation as variable modifications. The false discovery rate (FDR) was set to 2%.

[0104] Example 1: Depletion of mAb using FcγRIIIa affinity chromatography In this application, mAb1 (an IgG4) and mAb2 (an IgG1) were used as proof-of-concept samples. Therefore, their ability to be retained on an FcγRIIIa affinity column with and without Fc domain glycosylation was evaluated. Samples were loaded onto the FcγRIIIa affinity column after buffer exchange with mobile phase A. The gradient elution step was modified to a trap and elution method as described in the "Materials and Methods" section. Error! Reference source not found. A and 2B show that mAb1 and mAb2 were retained on the FcγRIIIa affinity column in their native (dashed trace) and deglycosylated (solid trace) forms. The deglycosylated mAb showed only low affinity for binding to the column and eluting in the FT fraction.

[0105] The suitability of this method for analyzing HCP based on mAb-based therapeutic fragments was also evaluated. To produce smaller fragments, mAb2 was digested using the enzymes IdeS and FabALACTICA, which cleave the mAb above and below the hinge region. Subsequently, the ability of FcγRIIIa resin to retain smaller fragments with Fc domain glycosylation was evaluated. Figure 3 and 4 ).

[0106] As shown in A, FabALACTICA can simultaneously produce Fab and Fc fragments. The FcγRIIIa affinity chromatogram of the digest shows two peaks: one as the FT fraction eluted at retention time (RT) 8.3 min (collected as fraction 1), and the other as a multiplet eluted at RT 30–45 min (collected as fraction 2) (A). The two collected fractions were concentrated using a 10 kDa filter and analyzed by RP-LC-MS. The total ion current (TIC) of each fraction is shown in B and C. The deconvolution mass spectra of the TIC show the expected molecular weight of the detected fragment (D and 3E). The results confirm that the Fab fragment showed no affinity for the FcγRIIIa affinity column and subsequently eluted in the FT fraction.

[0107] IdeS cleaves mAb at a single site below the hinge region, producing F(ab')2 and Fc / 2 fragments, as shown in A. Surprisingly, the FcγRIIIa chromatogram of mAb1 digested with IdeS shows a single peak, eluting as an FT fraction at RT 8.0 min. RP-LC-MS analysis of this fraction showed two well-separated peaks (RT 14.8 and 16.8 min). Deconvolution MS spectra of each peak confirmed the presence of the Fc / 2 and F(ab)'2 fragments, as shown in C and 4D, respectively.

[0108] A single peak was present in the chromatogram of FcγRIIIa. Subsequently, the Fc / 2 fragment of the collected fraction was identified by RP-LC-MS, indicating that the Fc / 2 fragment has no binding affinity or at least low binding affinity to FcγRIIIa resin and was eluted in the FT fraction.

[0109] These results indicate that Fc receptors immobilized on FcγRIIIa columns have a low affinity for binding to smaller mAb fragments (e.g., Fc / 2) containing glycosylated Fc domains. Optimal results were obtained using antibodies containing full-length Fc domains (e.g., IgG isotype antibodies). These results also demonstrate how to identify and select Fc affinity resins, i.e., Fc receptors, to match selected and given recombinant proteins, thereby enabling the selected and given recombinant proteins to bind efficiently to and be retained by the Fc resin, while any protein contaminants are separated from the recombinant proteins bound to the resin and can be collected in the flow-through for analysis. The interaction profiles of different types of Fc receptors are known to those skilled in the art. For example, Russell et al. discussed the binding profiles of various types of Fc receptors

[13] . Currently, the vast majority of approved mAb therapies belong to the IgG1 and IgG4 subclasses. Therefore, the disclosed methods have broad applicability for HCP characterization in commercially relevant antibody products.

[0110] Example 2: Rapid digestion for high-throughput HCP analysis The digestion procedure of this invention offers advantages such as short processing time, high efficiency, and high throughput. Sample loss can be a major problem in HCP proteomics analysis, as only trace amounts are typically present in the final product. Therefore, given that HCP identification and quantification require at least 2 to 3 unique peptides, applying conventional classical sample processing workflows is challenging. To evaluate the quality of the digestion procedure of this method for identifying low-level HCPs, UPS1 was reduced and digested for up to 60 minutes according to the procedure described in "Materials and Methods," with measurements taken at 15-minute intervals. UPS1 contained a mixture of 48 proteins with equimolar concentrations and molecular weights (MW) ranging from 6.3 to 82.9 kDa. To further investigate the quality of the digestion, the average protein sequence coverage, the number of identified proteins, and the sum of protein signal intensities at each time point were compared. Figure 5 ).

[0111] Error! Citation source not found. The total signal intensity (shown as "Total Peptide MS Intensity") and average sequence coverage (shown as percentages above each of the four columns) are summarized for each digestion condition studied. A 15-minute digestion time resulted in the identification of 47 out of 48 UPS1 proteins (the number identified is the height of the corresponding column, scaled as "Number of Proteins Detected" on the left). Four different shades in each column indicate the percentage of sequence coverage: with 15 minutes of digestion, most proteins were identified with an average sequence coverage of 45%. Increasing the digestion time to up to 45 minutes increased the average sequence coverage from 42% to 64%, and the signal intensity identification also increased (Error! Citation source not found.).

[0112] Compared to digestions of 30 and 45 minutes, a slight decrease in sequence coverage and peptide MS intensity was observed after 60 minutes of digestion. This indicates that the percentage of fully cleaved peptides (in cases where the peptide has more than one cleavage site) increases with increasing digestion time. This results in both very short peptides not preserved by the RP column and individually fully cleaved peptide fragments, which are not only well preserved by the RP column but also provide better and more reliable identification and quantification. Therefore, 60 minutes of digestion improves overall throughput in terms of digestion completeness while also increasing the intensity of uncut peptides. This method is expected to be robust, low-cost, and improve the throughput of HCP analysis.

[0113] Example 3: HCP analysis of FcγRIIIa affinity chromatographic fractions A semi-preparative FcγRIIIa affinity column was used because its large sample loading capacity allows for the injection of higher volumes of sample into the column, and sufficient material for proteomics analysis can be collected in a single run. Following the procedures outlined in "Materials and Methods," 5 mg of mAb1 sample was buffer-exchanged with FcγRIIIa loading buffer (mobile phase A) and loaded onto the FcγRIIIa affinity column. The FT fraction (fraction 1) was collected, concentrated, and the protein precipitated with acetone. The protein was then digested using a trypsin / Lys-C mixture and analyzed using LC-MS / MS as described in "Materials and Methods."

[0114] Assuming the sample loaded onto the FcγRIIIa column contains 200 ppm HCP, calculate the protein / enzyme ratio. This latter value can be adjusted to suit samples from different purification processes. HCP identification is based on the identification of at least one unique peptide having at least five amino acid residues.

[0115] For data analysis, a strategy similar to that recently reported [3] was adopted with minor adjustments, as described below. Peptide identification was extended to the full-scan MS level only when the peptide was detected by MS / MS in other fractions of the same mAb. These “computer-simulated peptides” were re-evaluated using the following criteria: retention time difference in the same sequence less than 30 seconds, quality accuracy of peptide quality of 5 ppm, MS1 correlation of 0.90, and signal intensity ≥ 5 x 10⁻⁶. 4 Furthermore, the experimental isotopic patterns of the identified peptides are in good agreement with the theoretical average distribution.

[0116] A total of 63 HCPs with at least one unique peptide were identified. A summary of the HCPs discovered by the FcγRIIIa affinity column is shown in Table 1.

[0117] The main HCPs identified (with at least 15% sequence coverage) were phospholipase B-like protein (PLBL2), protein S100, CC motif chemokine, glutathione S-transferase P, and heat shock homologous 71 kDa protein. The highest signal intensity of the identified HCPs corresponded to PLBL2, with a sequence coverage of 72.5%.

[0118] In addition to PLBL2, light chain (LC) and heavy chain (HC) with amino acid sequence coverage of over 90% were also identified. Although the FcγRIIIa column effectively depleted mAb1, product-related impurities such as fragments (e.g., LC or HC without Fc domains) did not bind to the column and were eluted into the FT fraction (HCP-rich fraction).

[0119] Table 1: HCPs identified in mAb 1 after depletion of protein A or FcγRIIIa affinity columns. Example 4: HCP analysis of separation fractions from protein A affinity chromatography: comparison with FcγRIIIa affinity chromatography Protein A affinity chromatography is frequently used as a general method for depleting mAbs prior to proteomics studies of HCPs[3]. Protein A-based separation relies on the strong affinity of the Fc region of the mAb to the protein A resin under neutral pH conditions, while most HCPs and other impurities are eluted in the FT fraction. Despite the excellent ability of protein A to deplete mAbs, non-specific interactions between the chromatographic resin and the mAb with certain HCPs reduce the practicality of this method in proteomics workflows. HCP-mAb interactions can involve a variety of forces, including electrostatic interactions, hydrogen bonds, hydrophobic interactions, and / or van der Waals forces. These interactions can prevent protein A chromatography from completely removing several types of HCPs, as the HCPs elute along with the mAb. To disrupt these interactions, pre-elution washes are typically applied in protein A chromatography prior to eluting the mAb. The effectiveness of pre-elution washes should be evaluated for each mAb to improve HCP recoveries prior to proteomics analysis and to prevent certain levels of risk when used in root cause investigations[3]. However, when rapid turnaround is required, product-specific pre-washing optimization is often a major bottleneck for biopharmaceutical companies.

[0120] Both FcγRIIIa and protein A resins can be used for affinity chromatography; however, for FcγRIIIa chromatography, nonspecific interactions that may be comparable to protein A-based purification methods have not been investigated. Therefore, the inventors investigated the efficiency of separating HCPs using FcγRIIIa and protein A chromatography.

[0121] For this purpose, following a recently described procedure, 5 mg of mAb1 was injected into a protein A affinity column [3]. The FT fraction (referred to as “F1-protein A”) was collected, and the column was washed with water. Pre-elution was performed in the presence of high salt concentration and alkaline pH (referred to as “F2-protein A”) [3]. Finally, mAb1 was eluted from the column using glycine buffer at pH 2.80 (referred to as “F3-protein A”). Figure 8 A schematic diagram of the protein A workflow is shown.

[0122] The FT and pre-eluting fractions (F1-protein A and F2-protein A) collected during protein A affinity chromatography were concentrated, precipitated, and digested, as described in "Materials and Methods," which is similar to the treatment of FT from FcγRIIIa chromatography.

[0123] Comparing the protein identification results for each method, the protocol using protein A produced 57 identified HCPs, while the Fc receptor-based method of the present invention produced 63 proteins in Example 3.

[0124] Similar to the Fc receptor approach, PLBL2 was detected in the protein A fractions (F1-protein A and F2-protein A) with sequence coverage exceeding 70%. PLBL2 is known as the "Hitchhiker HCP" because it is difficult to remove even after several chromatographic steps and may trigger an immune response in patients. To better understand HCP-mAb interactions, we evaluated the levels of PLBL2 in the FcγRIIIa and protein A affinity chromatographic fractions.

[0125] First, the MS peak areas of all PLBL2 peptides detected in the protein A fractions (F1-protein A, F2-protein A, and F3-protein A) were calculated. The data revealed that F2-protein A and F3-protein A had the highest and lowest signal intensities, respectively, at 93.9% (total MS signal: 3.8 x 10⁻⁶). 9 ) and 0.6% (total MS signal: 2.6 x 10 7 () Figure 7 A). 5.5% of the total PLBL2 signal was detected in F1-protein A (total MS signal: 2.2 x 10⁻⁶). 8 This demonstrates the relevance of the pre-elution step. This is particularly critical if only the F1-protein A fraction is used for proteomics studies, as most HCPs exist in trace amounts, and inadequate elution can result in concentrations below the detection limit of the MS method. Furthermore, the PLBL2 level in the protein A eluent (F3-protein A) depends on several parameters, including mAb subclass (IgG4 has a higher PLBL2 level in the eluent than IgG1), the PLBL2 concentration in the chromatographic load, the resin loading density, and the pre-elution washing conditions.

[0126] The PLBL2 level in fractions collected from FcγRIIIa chromatography was investigated. The main measured signal intensity was in F1 (99.6%, MS signal: 5.6 x 10⁻⁶). 9 This indicates that PLBL2 can be efficiently separated using FcγRIIIa chromatography in a single step. Error! Reference source not found. A and 7B show detailed information on the PLBL2 signal intensity detected by protein A and FcγRIIIa chromatography, respectively.

[0127] Based on the findings of this invention, a novel FcγRIIIa-based method for mAb depletion has been discovered to be more efficient than protein A chromatography. Most approved therapeutic mAbs are antibodies containing constant regions of IgG1 or IgG4 types, which can be easily depleted using Fc receptor-based chromatography (e.g., FcγRIIIa chromatography) without any significant optimization. The time required for sample preparation and data analysis is reduced due to the separation of HCPs in a single fraction. This accelerates process optimization. The comprehensive information obtained using the workflow disclosed herein provides invaluable insights for the HCP identification of approved therapeutic mAbs, assisting in the development of biosimilars.

[0128] References [1] M. Kiyoshi, JMM Caaveiro, M. Tada, H. Tamura, T. Tanaka, Y.Terao, K. Morante, A. Harazono, N. Hashii, H. Shibata, D. Kuroda, S.Nagatoishi, S. Oe, T. Ide, K. Tsumoto, A. Ishii-Watabe, Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineeredFcgammaReceptor IIIa-Immobilized Column, Sci Rep, 8 (2018) 3955. [2] M. Hiranyakorn, S. Iwamoto, A. Hoshinoo, R. Tsumura, H.Takashima, M. Yasunaga, S. Manabe, Chromatographic Analysis of the N-GlycanProfile on Therapeutic Antibodies Using FcgammaRIIIa Affinity ColumnChromatography, ACS Omega, 8 (2023) 16513-16518. [3] W. Esser-Skala, M. Segl, T. Wohlschlager, V. Reisinger, J. Holzmann, C.G. Huber, Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins, Anal Bioanal Chem, 412 (2020) 6583-6593. [4] L. Jiang, L. He, M. Fountoulakis, Comparison of protein precipitation methods for sample preparation prior to proteomic analysis, J Chromatogr A, 1023 (2004) 317-320. [5] M. Zarei, J. Jonveaux, P. Wang, F.M. Haller, B. Gu, A.V. Koulov, M. Jahn, Proteomic Analysis of Adenovirus 5 by UHPLC-MS / MS: Development of a Robust and Reproducible Sample Preparation Workflow, ACS Omega, 7 (2022) 36825-36835. [6] Kabat E.A. and Wu, T.T. (1991) J. Immunol., 147, 1709-1719. [7] Hawkins et al. J. Mol. Biol. 254, 889-896 (1992). [8] Lowman et al., Biochemistry 30, 10832-10837 (1991). [9] Kontermann and Dübel, Antibody Engineering, Springer, 2nd Edition, 2010.

[10] Kontermann 2015, Roland and Brinkmann, Ulrich. (2015). BispecificAntibodies, Drug Discovery Today 20.

[11] Edelman GM et al. Proc Natl Acad Sci USA. 1969; 63:78-85.

[12] Chakrabarti, A. et al., "Analytical Characterization of Monoclonal Antibodies with Novel Fc Receptor-Based Chromatography Technique", in Rezaei N (ed.) (2021) Monoclonal Antibodies. IntechOpen, ISBN 978-1-83968-370-1.

[13] Russell, A. et al., Int. J. Mol. Sci. 2018, 19, 390; doi:10.3390 / ijms19020390.

Claims

1. A method for separating and analyzing one or more protein contaminants in a sample containing recombinant proteins and one or more protein contaminants, the method comprising: i. Fractionation of the sample containing the recombinant protein and one or more protein contaminants, wherein the fractionation comprises contacting the sample with an Fc receptor affinity resin and collecting the flow-through to obtain a fraction containing one or more protein contaminants; ii. Analysis of the one or more protein contaminants in the fraction containing the one or more protein contaminants.

2. The method of claim 1, wherein the analysis in step b) comprises subjecting the fraction containing the one or more protein contaminants to concentration of the one or more protein contaminants.

3. The method of claim 2, wherein the concentration of said one or more protein contaminants comprises i. Precipitation of one or more protein contaminants; and ii. Dissolution of one or more precipitated protein contaminants in a suitable solvent.

4. The method according to any one of claims 1 to 3, wherein the analysis in step b) comprises the fragmentation of the one or more protein contaminants.

5. The method of claim 4, wherein the method comprises, before or after the fragmentation, preferably before the fragmentation, the chemical reduction of the one or more protein contaminants.

6. The method according to claims 1 to 5, wherein the analysis of the one or more protein contaminants in step b) includes the identification of one or more individual protein contaminants, preferably peptide analysis by liquid chromatography-mass spectrometry (LC-MS).

7. The method according to any one of claims 1 to 6, wherein the Fc receptor affinity resin comprises an immobilized Fc receptor that binds to the recombinant protein.

8. The method according to any one of claims 1 to 7, wherein the Fc receptor affinity resin is an Fcγ receptor affinity resin or a neonatal Fc receptor (FcRn) affinity resin, preferably an Fcγ receptor affinity resin.

9. The method according to claim 8, wherein the Fcγ receptor affinity resin is a type I Fcγ receptor (FcγR) affinity resin, a type II Fcγ receptor (FcγR) affinity resin, or a type III Fcγ receptor (FcγR) affinity resin; preferably, the Fcγ receptor affinity resin is a type I Fcγ receptor (FcγR) affinity resin; more preferably, the type I Fcγ receptor (FcγR) affinity resin is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb affinity resins; and even more preferably, the type I Fcγ receptor (FcγR) affinity resin is an FcγRIIIa affinity resin.

10. The method according to any one of claims 1 to 9, wherein the recombinant protein binds to the Fc receptor affinity resin.

11. The method according to any one of claims 1 to 10, wherein the recombinant protein comprises an Fc domain or a fragment of an Fc domain, preferably a glycosylated Fc domain or a fragment of a glycosylated Fc domain.

12. The method according to any one of claims 1 to 11, wherein the recombinant protein is an antibody or its antigen-binding fragment or Fc fusion protein.

13. The method according to any one of claims 1 to 12, wherein the recombinant protein comprises an IgG constant region; preferably, the IgG constant region is an IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region; more preferably, the IgG constant region is an IgG1 constant region, an IgG2 constant region, or an IgG4 constant region; even more preferably, the IgG constant region is an IgG1 constant region or an IgG4 constant region.

14. The method according to any one of claims 1 to 13, wherein the recombinant protein is an IgG antibody, preferably an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody, more preferably an IgG1 antibody, an IgG2 antibody or an IgG4 antibody, and even more preferably an IgG1 antibody or an IgG4 antibody.

15. The method according to any one of claims 1 to 14, wherein the recombinant protein is glycosylated, preferably wherein the recombinant protein comprises N-glycan.

16. The method of claim 15, wherein the N-glycan is an oligosaccharide having 3 to 20, preferably 3 to 15, particularly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 sugar residues.

17. The method of claim 3, wherein the precipitation of the one or more protein contaminants comprises the addition of a precipitant; preferably, the precipitant is selected from the group consisting of trichloroacetic acid (TCA), acetone, ammonium sulfate, chloroform, methanol, and water, and a combination of chloroform and methanol, preferably acetone.

18. The method of claim 17, wherein the precipitation of the one or more protein contaminants comprises the addition of the precipitant in an amount of at least about 2:1 relative to the volume of the fraction containing the one or more protein contaminants; and / or the precipitation of the one or more protein contaminants comprises the addition of a saline solution; preferably, the addition of the saline solution is performed prior to the addition of the precipitant; more preferably, the saline solution is an aqueous solution of a salt selected from the group consisting of NaCl.

19. The method according to any one of claims 3, 17 or 18, wherein the suitable solvent is an aqueous buffer containing a detergent, preferably selected from the group consisting of sodium deoxycholate, N-dodecyl-β-D-maltodextrin and combinations thereof.

20. The method of claim 4, wherein the fragmentation of the one or more protein contaminants is accomplished by proteolytic digestion; preferably, the proteolytic digestion comprises using a protease selected from the group consisting of trypsin, chymotrypsin, ArgC, GluC, AspN, LysC, LysN, elastase, and proteinase K and combinations thereof; more preferably trypsin and LysC.

21. The method according to any one of claims 4 or 20, wherein the protein hydrolysis is performed at a temperature between about 34°C and about 40°C; and / or wherein the protein hydrolysis comprises using one or more proteases at a ratio between about 1:5 and 1:40 (w / w, protease to protein substrate); and / or allowing the protein hydrolysis to proceed for at least about 15 minutes; and / or allowing the protein hydrolysis to proceed for a time between about 30 minutes and about 60 minutes.

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